Our research effort is directed toward the elucidation of the normal pathways for biosynthesis of pancreatic glucagon. This peptide, like insulin appears to be synthesized initially via a larger (precursor) peptide which is enzymatically cleaved to yield its biologically active product prior to secretion from the cells of origin. Using a subcellular fractionation procedure in conjunction with selective incorporation of isotopically labeled amino acids into the peptides of interest we have succeeded in identifying the intracellular sites for synthesis packaging and storage of glucagon. We are currently engaged in studies designed to elucidate the subcellular site(s) for, and the mechanism of, proteolytic processing of proinsulin and proglucagon by employing specific inhibitors of proteolysis in the presence of pre-labeled precursors in subcellular fractions. Subcellular fractions will also be assayed for their content of specific proteolytic enzymes. A separate portion of our work is directed toward the isolation and chemical characterization of the precursors and their various cleavage intermediates.